1. Field of the Invention
7-ethyl Camptothecin ("ECPT") is a potent inhibitor of the enzyme Topoisomerase I and has demonstrated broad anticancer activity in a variety of preclinical tumor models. The lactone form of Camptothecin (CPT) is poorly soluble in water and has significant antitumor activity and hydrolysis of E-ring lactone to the carboxylate form of camptothecin greatly increases the water solubility of molecule at the expense of significantly reducing its antitumor activity. A lactone stable form of camptothecin has not been administered by parenteral or oral routes in humans for the purpose of inhibiting the growth of cancer cells. This invention overcomes these limitations and claims novel pharmaceutically acceptable formulations of lactone stable ECPT, methods of administration of lactone stable ECPT, and antitumor compositions comprising solutions of lactone stable ECPT. Additionally, this invention claims novel dosages, schedules of administration, and routes of administration of ECPT formulations to humans with various forms of cancer.
2. Description of the Related Art
Introduction
A. DNA Topoisomerases
Several clinically important anticancer drugs kill tumor cells by affecting DNA topoisomerases. Topoisomerases are essential nuclear enzymes that function in DNA replication and tertiary structural modifications, such as overwinding, underwinding, and catenation, which normally arise during replication, transcription, and perhaps other DNA processes. Two major topoisomerases that are ubiquitous to all eukaryotic cells: (1) Topoisomerase I (topo I) which cleaves single stranded DNA; and (2) Topoisomerase II (topo II) which cleaves double stranded DNA. Topoisomerase I is involved in DNA replication; it relieves the torsional strain introduced ahead of the moving replication fork.
Topoisomerase I purified from human colon carcinoma cells or calf thymus has been shown to be inhibited by (a) camptothecin, (b) a water soluble analog called "CPT-11," and (c) 10-hydroxy 7-ethyl camptothecin (HECPT) which is the proposed active metabolite of CPT-11. CPT-11, camptothecin, and an additional Topo I inhibitor, topotecan, has been in used in clinical trials to treat certain types of human cancer. For the purpose of this invention, camptothecin derivatives include 7-ethyl camptothecin (ECPT), CPT-11, 10-hydroxy 7-ethyl camptothecin (HECPT), 9-amino camptothecin, 10,11 methylenedioxy camptothecin and topotecan. These camptothecin derivatives use the same mechanism to inhibit Topo I; they stabilize the covalent complex of enzyme and strand-cleaved DNA, which is an intermediate in the catalytic mechanism. These compounds have no binding affinity for either isolated DNA or topoisomerase I but do bind with measurable affinity to the enzyme-DNA complex. The stabilization of the topoisomerase I "cleavable complex" by camptothecin, CPT-11, or HECPT is readily reversible.
Although camptothecin and the aforementioned camptothecin derivatives have no effect on topoisomerase II, these camptothecin derivatives stabilize the "cleavable complex" in a manner analogous to the way in which epipodophyllotoxin glycosides and various anthracyclines inhibit topoisomerase II.
Inhibition of topoisomerase I by camptothecin and derivatives induces protein-associated-DNA single-strand breaks. Virtually all of the DNA strand breaks observed in vitro cells treated with camptothecin and derivatives are protein linked. However, an increase in unexplained protein-free breaks can be detected in L1210 cells treated with camptothecin. The compounds appear to produce identical DNA cleavage patterns in end-labeled linear DNA. It has not been demonstrated that camptothecin or active derivatives of camptothecin cleaves DNA in the absence of the topoisomerase I enzyme.
B. Activity of Camptothecin and Derivatives is Cell Cycle Specific
The activity of camptothecin and active camptothecin derivatives is cell cycle specific. The greatest quantitative biochemical effect observed in cells exposed to HECPT is DNA single-strand breaks that occur during the S-phase. Because the S-phase is a relatively short phase of the cell cycle, longer exposure to the drugs results in increased cell killing. Brief exposure of tumor cells to the drugs produces little or no cell killing, and quiescent cells are refractory. These results are likely due to two factors:
(1) The drugs inhibit topoisomerase I reversibly. Although they may produce potentially lethal modifications of the DNA structure during DNA replication, the breaks may be repaired after washout of the drug; and PA1 (2) Cells treated with topo I inhibitors, such as camptothecin, tend to stay in G0 of the cell cycle until the drug is removed and the cleaved DNA is repaired. Inhibitors of these enzymes can affect many aspects of cell metabolism including replication, transcription, recombination, and chromosomal segregation. PA1 (1) direct administration of ECPT allows the clinician to tailor the administration of the active cytoxic species (lactone stable ECPT) to suit the patient's tolerance; PA1 (2) direct administration of ECPT overcomes interpatient variability which may be due to polymorphism of key enzyme(s) in the metabolism of CPT-11 to HECPT; and PA1 (3) clinicians can more consistently optimize the drug dosage and schedule to achieve the maximum tolerated dose of ECPT which is likely to lead to the most beneficial clinical anti-cancer effect. PA1 (1) methods of administering lactone stable ECPT to patients with cancer; PA1 (2) solutions of lactone stable ECPT; PA1 (3) antitumor compositions comprising lactone stable ECPT; PA1 (4) stable formulations of lactone stable ECPT suitable for parenteral administration; PA1 (5) pharmacologic schedules for achieving the maximum tolerated dose with acceptable clinical toxicity observed in standard clinical practice of cancer treatment; PA1 (6) a novel oral formulation of ECPT; and PA1 (7) use of ECPT for the treatment of localized complications of cancer by direct administration via instillation into various body cavities.
C. Lactone Form Stabilizes 7-Ethyl Camptothecin Antitumor Activity and Reduces Water Solubility
Utilizing HPLC and NMR techniques, it has been demonstrated that camptothecin and camptothecin derivatives with native lactone E-ring moieties undergo an alkaline, pH-dependent hydrolysis of the E-ring lactone. The slow reaction kinetics allows one to assess if both the lactone and non-lactone forms of the drug stabilizes the topoisomerase I-cleaved DNA complex. Studies indicate that only the closed lactone form of the drug helps stabilize the cleavable complex. This observation provides reasoning for the high degree of camptothecin activity observed in solid tumor models. Tumor cells, particularly hypoxic cells prevalent in solid neoplasms, have lower intracellular pH levels than normal cells. At pH levels below 7.0, the closed form of camptothecin predominates. Thus, the inventors predict that ECPT will be more effective at inhibiting topoisomerase I in an acidic environment than in cells having higher intracellular pH levels. This invention provides lactone stable ECPT as the basis of the claimed subject matter. For this invention, lactone stable ECPT is defined as ECPT which is dissolved in DMI or DMA in the presence of a pharmaceutically acceptable acid. The presence of the acid stabilizes the lactone form of ECPT. For the purpose of this invention lactone stable ECPT and ECPT are used interchangeably.
D. Camptothecin and Derivatives
In 1966, Wall and Wani isolated camptothecin from the plant, Camptotheca acuminata. In the early 1970's, camptothecin reached Phase I trials and was found to have antitumor activity, but it caused unpredictable myelosuppression and hemorrhagic cystitis. Phase II studies with sodium camptothecin were limited because they induced unpredictable and severe myelosuppression, gastrointestinal toxicity, hemorrhagic cystitis, and alopecia. Clinical trials with sodium camptothecin were eventually discontinued because of unpredictable toxicities.
Because of these limitations and the fact that it is poorly soluble in water, camptothecin has been considered unsuitable for direct clinical use. One aspect of this invention is to formulate ECPT in a pharmaceutically acceptable manner using an organic solvent or a mixture of organic co-solvents to stabilize CPT in the lactone ring form. It is this lactone stable ECPT which permits direct administration of CPT to cancer patients. An additional embodiment of this invention is to provide specific indications, schedules, dosages and routes of administration of lactone stable ECPT for the purpose of treating cancer in humans.
The selection of suitable organic solvents for pharmaceutical dosage forms is limited to those which have a high degree of physiological safety. This invention describes administration of lactone stable ECPT in a pharmaceutically acceptable multi-solvent formulation, overcomes interpatient variability and drug resistance as it relates to the CPT-11 conversion to HECPT and is useful in instances where human cancer cells, because of their altered enzymatic activity, resist metabolic conversion of CPT-11 to HECPT.
Two camptothecin derivatives, CPT-11 and topotecan, have less sporadic toxicities but retain significant activity of the parent compound. CPT-11 and topotecan are currently undergoing Phase I and Phase II development in the United States. 10,11 methylene dioxycamptothecin is reportedly very active in preclinical studies, but it is also reported to be relatively insoluble in water which limits its use in the clinic (Pommier, et al. 1992).
In preclinical studies, Kunimoto and co-workers administered camptothecin at similar dosages (10-100 mg/kg intraperitoneally) to CDF1 mice implanted with intraperitoneal L1210 leukemia and demonstrated superior T/C (treated/control) ratios relative to mice treated in the same manner with 7-ethyl camptothecin (ECPT) and 10-hydroxy 7-ethyl camptothecin (HECPT). Their results with camptothecin, ECPT and HECPT were inferior that of CPT-11 administration under the same conditions. The inventors of the instant invention believe that the lesser activity observed by Kunimoto is related to the lack of an optimized pharmacologic schedule for ECPT. The instant invention takes into account the requirement for administration of the lactone stable species of ECPT by a prolonged, not bolus, parenteral infusion or by the repeated oral, parenteral or topical administration of the drug in a manner which closely replicates the pharmacokinetics of a continuous parenteral infusion.
Tables 1 and 2 present data summarizing Phase I and Phase II clinical trials of CPT-11. Neutropenia and diarrhea are the major reported, dose-limiting toxicities of CPT-11.
TABLE 1 __________________________________________________________________________ PHASE I STUDES CPT-11 Investigator Schedule # Pts Dose Toxicity Tumor Type __________________________________________________________________________ Clavel et al 90 min. 37 pts 115 mg/m.sup.2 /d Neutropenia* diarrhea, Breast (1 PR) QDx 3 Q21 days (33-115) nausea and vomiting, Mesothelioma alopecia (1 PR) Culine et al 90 min. 59 150 mg/m.sup.2 /wk Neutropenia* diarrhea* esophagus (1 PR) Q21 days (50-150) vomiting, alopecia cervix (1 PR) fatigue stomatitis renal (1 PR) Neutropenia* ovarian (1 PR) Negoro et al 30 min 17 100 mg/m.sup.2 Diarrhea*, N/V, NS CLC (2 PRs) infusion (50-150 alopecia, liver dys- function Ohe et al 120 hr CI 36 40 mg/m.sup.2 /d Diarrhea* nausea and None Q3 wks (5-40) vomiting, thrombo- cytopenia, anemia, liver dysfunction Diarrhea* Rothenberg et al 90 mg QWx 4 32 180 mg/m.sup.2 /wk Neutropenia, nausea, Colon Ca (2 PRs) Q42 days (50-180) vomiting, alopecia Rowinsky et al 90 min infusion 32 240 mg/m.sup.2 Neutropenia* vomiting, Colon Ca (1 PR) Q21 day (100-345) diarrhea abd. pain, Cervix Ca (1 PR) flushing __________________________________________________________________________ *Dose Limiting Toxicity
TABLE 2 __________________________________________________________________________ CPT-11 PHASE II TRIALS Investigator Tumor Type Schedule # Pts Response Rate Reported Toxicities __________________________________________________________________________ Fukuoka et al Untreated Non Small 100 mg/m.sup.2 weekday 73 (23/72) PRs Neutropenia diarrhea, Cell Lung Cancer 31.9% nausea, vomiting, anorexia, alopecia Masudu et al Refractory or Relapsed 100 mg/m.sup.2 weekly 16 (7/15) PRs Neutropenia, diarrhea Small Cell Lung Ca 47% pneumonitis (12.5%) Negoro et al Small Cell Lung Cancer 100 mg/m.sup.2 /week 41 2 CRs and 7 Neutropenia (38.6%) PRs 33.3% N/V (61.5%) diarrhea (53.8%) alopecia (40.0%) Ohno et al Leukemia/Lymphoma 200 mg Q3 No resp. 62 ** Neutropenia (91%) 40 mg/m.sup.2 Q0x5 34% PR Thrombocytopenia 20 mg/m.sup.2 bid x7 25% RR Gastrointestina 1 (76%) Shimada et al Colon cancer 100 mg/m.sup.2 /week or 17 6/17 (PR) Neutropenia (53%) 150 mg/m.sup.2 /Q 2 wks 46% NN (35%) diarrhea (24%) Takeuchi et al Cervical cancer 100 mg/m.sup.2 weekly 69 SCR Neutropenia (89%) 150 mg/m.sup.2 weeks 8 PR N/V (51%) RR of 23.6% Diarrhea (39.1%) Alopecia (38.1%) __________________________________________________________________________ **see text
E. HECPT is the Active Metabolite of CPT-11
Preclinical data, obtained by Barilero et al. on animals and more recently on humans, suggest that HECPT is the active metabolite of CPT-11 in vivo. Several different researchers administered CPT-11 and HECPT intravenously during Phase I trials and recorded the peak plasma concentrations (CpMax) at the end of the infusions. An analysis of the published mean peak plasma concentrations indicates that approximately 1.5% to 9% of the administered CPT-11 (on a per/mg basis) is converted into HECPT. The pharmacokinetic data from 30-minute intravenous infusions show a lower percentage of conversion (.about.1.5%) of CPT-11 to HECPT than that observed following more prolonged infusions (.about.9% at 40 mg/m.sup.2 /d.times.5). The reported half life of HECPT observed in humans following the administration of CPT-11 ranges from 8.8 to 39.0 hours.
The biochemical and pharmacological relationship between CPT-11 and HECPT, as well as the role these compounds play in killing cancer cells in vivo is not completely understood. Investigators studying in vitro tumor cell lines have reported that HECPT has a 3600-fold greater inhibitory activity than CPT-11 against topoisomerase I in P388 cells and that HECPT is approximately 1000-fold more potent in generating single-strand DNA breaks in MOLT3 cells (Kawato, et al ( 1991)). However, Kaneda et al. report that HECPT has little anti-tumor activity compared to CPT-11 in vivo. They base their findings on studies conducted using an intermittent bolus schedule (days 1, 5, and 9) and an i.p. route of administration with an intraperitoneal P388 tumor model in mice.
Ohe et al. suggest that HECPT is a more toxic moiety of CPT-11 and could be responsible for much of the toxicity attributed to CPT-11. However, these same investigators noted a lack of correlation between HECPT pharmacokinetics and dose or CPT-11 pharmacokinetics and toxicity in human subjects. Furthermore, Ohe et al. noted a large range of interpatient variability in the AUC of CPT-11 and its metabolism to HECPT, which may result in unpredictable variability in the pharmacokinetic behavior, clinical anti-tumor effects, and toxicity in the individual patient. The data Ohe et al. obtained (using a 5-day, continuous intravenous infusion of CPT-11) also suggests that the conversion of CPT-11 to HECPT is a saturable process. If this is so, the clinical approach to maximizing dose intensity of the active metabolite would impose additional limitations on the effective use of CPT-11.
In preclinical studies of xenografts of human tumors in nude mice, Kawato et al. report that the sensitivity of human tumors to CPT-11 is independent of their ability to produce HECPT and that the effectiveness of CPT-11 is not related to the ability of the tumor to produce HECPT. Kawato et al. suggests that HECPT production is likely to be mediated in the plasma or interstitial compartment. Kaneda et al. observed that the plasma concentration of HECPT in mice was maintained longer after CPT-11 administration than after treatment with HECPT and suggested that clinicians should maintain plasma levels of HECPT to enhance the antitumor activity of CPT-11. The present invention has a useful advantage of not requiring activation by an enzyme in order to form the active species (as with CPT-11) and the additional advantage of being able to directly control the interpatient variability.
One of the advantages of present invention provides clinicians with the ability to directly adjust the plasma levels of ECPT to the point of therapeutic tolerance by controlling the dose and the schedule of administration. The inventors contend that this should lead to a superior ability to achieve better antitumor activity and reduce interpatient variability of the plasma levels of ECPT.
The different observations made in these studies suggest that direct administration of ECPT by parenteral and oral administration could provide significant clinical benefit for the treatment of cancer. However, in the past, ECPT has been considered insufficiently water soluble for clinical use. The claimed invention overcomes the solubility problem by providing lactone stable pharmaceutically acceptable multisolvent formulations of ECPT for parenteral use and also oral ECPT formulations.